Gelatin particles, method for producing gelatin particles, gelatin particle-containing cell, and method for producing gelatin particle-containing cell

ABSTRACT

Disclosed herein are gelatin particles including gelatin, wherein when a major-axis length of dried gelatin particles is defined as a and a major-axis length of gelatin particles after swelling treatment obtained by immersing the dried gelatin particles in water at 40° C. under an atmospheric pressure for 60 minutes is defined as b, swelling degree represented by b/a is 1.0 or more but 10.0 or less, and wherein the gelatin particles after swelling treatment have a particle diameter of 1.0 nm or more but 5.0 μm or less. The gelatin particles are easily taken up by cells themselves.

TECHNICAL FIELD

The present invention relates to gelatin particles, a method forproducing gelatin particles, a gelatin particle-containing cell, and amethod for producing a gelatin particle-containing cell.

BACKGROUND ART

Gelatin is highly biocompatible and has the property of being degradedand readily absorbed by the body. Therefore, a technique has beendeveloped in which a substance such as a reagent or a drug (hereinafter,simply referred to as “reagent or the like”) encapsulated in gelatin inthe form of particles is delivered and released in the living body.

For example, Patent Literature 1 discloses solid spherical swellablegelatin particles made of thermally-crosslinked gelatin having a jellystrength of 80 to 120 g. The dried gelatin particles before swellinghave a particle diameter of 20 to 1600 μm, and the dried gelatinparticles after swelling have a particle diameter of 50 to 2000 μm.According to Patent Literature 1, the swellable gelatin particles haveexcellent shape retentivity and are hard to break even when deformed bythe application of external stress, and are therefore suitable forintravascular administration using a microcatheter or a syringe needle.

CITATION LIST Patent Literature

Patent Literature 1: JP 2014-58465 A

SUMMARY OF INVENTION Technical Problem

The gelatin particles disclosed in Patent Literature 1 are considered tobe suitable for use in so-called drug delivery system (DDS) in which thegelatin particles are administered into a blood vessel, an organ, or thelike to delivery and release a reagent or the like.

Meanwhile, in recent years, there is an increasing demand for thetechnique of directly introducing a reagent or the like into livingcells. For example, when a contrast medium is introduced into livingcells, the activity of the cells can be non-destructively examined.Further, when living cells having a contrast medium introduced thereintoare transplanted into a patient, it is possible to externally andless-invasively examine whether or not the transplanted cells have beencolonized without incising a transplantation site again. Since gelatinhas high biocompatibility, gelatin particles are considered to besuitable also as carriers to carry a reagent or the like to beintroduced into living cells.

An electroporation method or a microinjection method may be used tointroduce gelatin particles carrying a reagent or the like into livingcells. However, such a method is performed by changing the shape of thecell membrane to introduce a reagent or the like into the inside of thecell membrane, and therefore there is a fear that the cell membrane ispartially broken so that the activity of cells is reduced. From theviewpoint of minimizing such a reduction in the activity of cells, areagent or the like is preferably taken up into cells by cell's ownaction. Therefore, gelatin particles carrying a reagent or the like arealso preferably easily taken up into cells by cell's own action.However, according to the findings of the present inventors, the gelatinparticles disclosed in Patent Literature 1 are hard to be taken up intocells by cell's own action.

In light of the above problems, it is an object of the present inventionto provide gelatin particles that are easily taken up into cells bycell's own action, a method for producing such gelatin particles, a cellhaving such gelatin particles, and a method for producing a cell havingsuch gelatin particles.

Solution to Problem

In order to achieve the above object, the present invention provides thefollowing means.

-   -   [1] Gelatin particles, wherein when a major-axis length of dried        gelatin particles is defined as a and a major-axis length of        gelatin particles after swelling treatment obtained by immersing        the dried gelatin particles in water at 40° C. under an        atmospheric pressure for 60 minutes is defined as b, swelling        degree represented by b/a is 1.0 or more but 10.0 or less, and        wherein the gelatin particles after swelling treatment have a        particle diameter of 1.0 nm or more but 5.0 μm or less.    -   [2] The gelatin particles according to [1], wherein the dried        gelatin particles have an aspect ratio of 1.0 or more but 1.4 or        less.    -   [3] The gelatin particles according to [1] or [2], wherein the        major-axis length b of the gelatin particles after swelling        treatment is 2.0 μm or less.    -   [4] The gelatin particles according to any one of [1] to [3],        further including a contrast medium.    -   [5] The gelatin particles according to any one of [1] to [4],        wherein the gelatin has been crosslinked.    -   [6] A method for producing gelatin particles, including        discharging droplets of a liquid containing melted gelatin into        an atmosphere in a heating tube or a drying chamber whose        difference in temperature from the droplets is 235° C. or less        and drying the droplets to obtain gelatin particles.    -   [7] The method for producing gelatin particles according to [6],        wherein the droplets are discharged from a nozzle of an inkjet        head.    -   [8] A gelatin particle-containing cell, wherein the gelatin        particles according to any one of [1] to [5] are contained        inside a cell membrane.    -   [9] A method for producing a gelatin particle-containing cell,        including adding the gelatin particles according to any one of        [1] to [5] and a cell to a liquid to allow the gelatin particles        to be taken up inside a cell membrane of the cell by action of        the cell.

Advantageous Effects of Invention

According to the present invention, it is possible to provide gelatinparticles that are easily taken up by cells themselves, a method forproducing such gelatin particles, a cell having such gelatin particles,and a method for producing a cell having such gelatin particles.

DESCRIPTION OF EMBODIMENTS

In order to achieve the above object, the present inventors haveintensively studied conditions required of gelatin particles that areeasily taken up into cells by cell's own action. As a result, thepresent inventors have found that gelatin particles whose swellingdegree when absorbing water is low and whose particle diameter afterswelling in water is 1.0 nm or more but 5.0 μm or less are easily takenup into cells by cell's own action. This finding has led to thecompletion of the present invention.

Hereinbelow, exemplary embodiments of the present invention will bedescribed in detail.

1. Gelatin particles and method for producing the same

This embodiment relates to gelatin particles and a method for producinggelatin particles.

1-1. Gelatin particles

The gelatin particles according to this embodiment are gelatin particlesincluding gelatin, wherein when a major-axis length of dried gelatinparticles is defined as a and a major-axis length of gelatin particlesafter swelling treatment obtained by immersing the dried gelatinparticles in water at 40° C. under an atmospheric pressure for 60minutes is defined as b, swelling degree represented by b/a is 1 or morebut 10 or less, and wherein the gelatin particles after swellingtreatment have a particle diameter of 1.0 nm or more but 5.0 μm or less.As will be described later, gelatin particles having such a structure asdescribed above are easily taken up by cells, and are therefore alsoreferred to as “easy-uptake gelatin particles” in this description. Theeasy-uptake gelatin particles may be single particles or aggregates oftwo or more gelatin particles.

It is to be noted that in this description, the dried gelatin particlesrefer to gelatin particles dried by allowing them to stand in theatmosphere at 80° C. for 24 hours. Further, in this description, thegelatin particles after swelling treatment refer to gelatin particlesobtained by immersing dried gelatin particles in water at 40° C. underthe atmospheric pressure for 60 minutes.

The minor-axis length and major-axis length of the easy-uptake gelatinparticles may be values obtained by analyzing an image taken by ascanning electron microscope (SEM). When the gelatin particles are theabove-described aggregates, the major-axis length, minor-axis length,particle diameter, and aspect ratio of the gelatin particles may be theaverages of the major-axis lengths, minor-axis lengths, particlediameters, and aspect ratios of a plurality of gelatin particles afterswelling treatment (e.g., 20 gelatin particles) randomly selected fromthe aggregates, respectively.

The easy-uptake gelatin particles have a swelling degree of 1.0 or morebut 10.0 or less. If the swelling degree is larger than 10.0, thegelatin particles absorb a larger amount of water when swelling, and aretherefore likely to aggregate due to the action of absorbed water. It isconsidered that when many swelled gelatin particles aggregate, theapparent particle diameter of the gelatin particles increases, andtherefore the gelatin particles are likely to be recognized as foreignmatter by cells and are hard to be taken up into cells by cell's ownaction. On the other hand, the gelatin particles having a swellingdegree of 10.0 or less do not absorb so much water (i.e., have a lowswelling degree), and are therefore hard to aggregate even afterswelling, and the apparent particle diameter of the gelatin particles isless likely to increase. Therefore, it is considered that the gelatinparticles having a swelling degree of 10.0 or less are less likely to berecognized as foreign matter by cells even after swelling by absorptionof water, and are therefore easily taken up into cells by cell's ownaction. From the above viewpoint, the swelling degree is preferably 1.0or more but 8.0 or less, more preferably 1.0 or more but 5.0 or less.

The gelatin particles after swelling treatment have a particle diameterof 1.0 nm or more but 5.0 μm or less. The gelatin particles having aparticle diameter of 5.0 μm or less are easily taken up into cells bycell's own action. The reason for this is considered to be that thegelatin particles having a particle diameter of 5.0 μm or less are lesslikely to be recognized as foreign matter by cells, and are thereforeeasily taken up into cells by action such as endocytosis. From the aboveviewpoint, the particle diameter of the gelatin particles after swellingtreatment is preferably 2.0 μm or less, more preferably 1.5 μm or less.On the other hand, the gelatin particles having a particle diameter of1.0 nm or more can easily carry a reagent or the like therein. From theabove viewpoint, the particle diameter of the gelatin particles afterswelling treatment is preferably 2.0 nm or more. Further, when having aparticle diameter of 0.50 μm or more, the gelatin particles afterswelling treatment are excellent in handleability and can contain alarge amount of reagent or the like. It is to be noted that the particlediameter of the easy-uptake gelatin particles may be the average of themajor-axis length and the minor-axis length of the gelatin particles.

The dried gelatin particles preferably have an aspect ratio of 1.0 ormore but 1.4 or less. When the aspect ratio is 1.4 or less, the gelatinparticles are more likely to keep their nearly-spherical shape bothbefore and after swelling. Therefore, in a solution containing thegelatin particles and cells, the gelatin particles are likely to comeinto contact with the cells at contact surfaces having more uniformshape and size. From this, it is considered that there is littledifference in ease of uptake among the gelatin particles. Therefore, itis considered that when the easy-uptake gelatin particles have an aspectratio within the above range, the amount of the gelatin particles to betaken up into cells and the amount of cells that take up the gelatinparticles can be more easily controlled. The aspect ratio of theeasy-uptake gelatin particles may be a value determined by dividing themajor-axis length of the gelatin particles by the minor-axis length ofthe gelatin particles.

The major-axis length (b) of the gelatin particles after swellingtreatment is preferably 2.0 μm or less. It is considered that when themajor-axis length (b) is 2.0 μm or less, the gelatin particles are morelikely to keep their small particle diameter both before and afterswelling, and are therefore easily taken into cells by cell's ownaction. From the above viewpoint, the major-axis length (b) is morepreferably 1.8 μm or less, even more preferably 1.5 μm or less.

The easy-uptake gelatin particles are mainly made of gelatin. Morespecifically, the easy-uptake gelatin particles contain 300 or moreglycine residues out of 1000 amino acid residues and contain bothalanine and proline when analyzed with an amino acid analyzer. Thegelatin is not particularly limited as long as it is capable of formingparticles, and any known gelatin may be used which is obtained bydenaturing collagen derived from cattle bone, cattle skin, pig skin, pigtendon, fish scales, and fish meat. Gelatin has previously been used forfood and for medical purposes, and its intake into the body is hardlyharmful to the human body. Further, gelatin disperses and disappears inthe living body, and is therefore advantageous in that its removal fromthe living body is not required. It is to be noted that the easy-uptakegelatin particles may contain a component other than gelatin as long asthe gelatin particles can be taken up into cells. It is to be noted thatwhen the easy-uptake gelatin particles contain a component other thangelatin, the component is preferably contained to the extent that harmto the human body caused by intake into the body is negligible. Further,the component other than gelatin is preferably composed of a substancethat does not accumulate in the living body and is easily discharged.

From the viewpoint of easily forming gelatin particles that satisfy theabove-described conditions of particle diameter and swelling degree, theweight-average molecular weight of the gelatin constituting theeasy-uptake gelatin particles is preferably 1000 or more but 100000 orless. The weight-average molecular weight may be a value measured inaccordance with, for example, the PAGI Method Ver. 10 (2006).

The gelatin constituting the easy-uptake gelatin particles may becrosslinked. The crosslinking may be crosslinking achieved by using acrosslinking agent or self-crosslinking achieved without using acrosslinking agent.

The crosslinking agent may be, for example, a compound having aplurality of functional groups that form chemical bonds with a hydroxylgroup, a carboxyl group, an amino group, a thiol group, an imidazolegroup, or the like. Examples of such a crosslinking agent includeglutaraldehyde, water-soluble carbodiimides such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate(CMC), compounds having two or more epoxy groups such as ethylene glycoldiglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerolpolyglycidyl ether, and glycerol polyglycidyl ether, and propyleneoxide. Among them, from the viewpoint of further enhancing reactivity,glutaraldehyde and EDC are preferred, and glutaraldehyde is morepreferred.

Examples of the self-crosslinking include crosslinking achieved byapplication of heat and crosslinking achieved by irradiation withelectron beams or ultraviolet rays.

The easy-uptake gelatin particles may carry a reagent or the like. Thephrase “gelatin particles carry a reagent or the like” means that areagent or the like is present on the surfaces of the gelatin particlesor inside the gelatin particles. From the viewpoint of retaining thereagent or the like in cells for a longer period of time, the reagent orthe like is preferably present inside the gelatin particles.

Examples of the reagent or the like include: reagents to be used fortests of biological activity, measurements of substances in the livingbody, and quantitative analysis of substances in the living body; anddrugs. Examples of the reagents include contrast media.

Examples of the contrast media include magnetic substances for use ascontrast media for MRI. Examples of the contrast media for MRI includecontrast media containing gadolinium (Gd) and contrast media containingiron (e.g., Fe₃O₄ and γ-Fe₂O₃).

The drugs are not particularly limited as long as they can be carried bythe gelatin particles. Examples of such drugs include proteins havingpharmaceutical activity, plasmids, aptamers, antisense nucleic acids,ribozymes, nucleic acids used for pharmaceutical purposes, includingtRNA, snRNA, siRNA, shRNA, ncRNA, and condensed DNA, and antigens usedfor pharmaceutical purposes.

Examples of the proteins having pharmaceutical activity includesteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), vitamin A(retinoid), vitamin D3 and vitamin D3 analogs, antibiotics, antiviraldrugs, and antibacterial drugs.

1-2. Method for Producing Gelatin Particles

The easy-uptake gelatin particles can be produced by a known method inwhich gelatin is formed into particles. Examples of such a methodinclude: a method in which droplets of a liquid containing meltedgelatin (hereinafter, also simply referred to as “gelatin solution”) aredischarged into an atmosphere in a heating tube or a drying chamber anddried (in-air dropping method); a method in which droplets of a gelatinsolution are discharged into a hydrophobic solvent and dispersed(in-liquid dropping method); and a method in which a gelatin solution isemulsified to disperse microdroplets containing gelatin (in-liquiddispersion method). Examples of the in-air dropping method include aninkjet method and a spray drying method. Examples of the in-liquiddispersion method include an emulsion method and a coacervation method.From the viewpoint of producing gelatin particles having a more uniformparticle diameter and a smaller aspect ratio, an in-air dropping methodis preferred, and an inkjet method is more preferred.

According to new findings by the present inventors, an in-air droppingmethod is particularly preferred in which the above-described dropletsare dried under conditions where a temperature change is small. It isconsidered that this makes it possible to prevent gelatin particles frombeing deformed or disintegrated due to temperature change so thatgelatin particles having a particle diameter of 1.0 nm or more but 5.0μm or less are more likely to be produced and the produced gelatinparticles can have a more uniform particle diameter.

For example, in the case of an inkjet method, droplets of a gelatinsolution can be discharged from an inkjet nozzle provided inside aheated heating tube and collected by a filter provided inside the sameheating tube. From the viewpoint of further reducing deformation ordisintegration of gelatin particles, the heating of the heating tube ispreferably performed by allowing hot air to flow through the inside ofthe heating tube in the same direction as a direction in which thedroplets are dropped (i.e., in a vertical direction from the top towardthe bottom of the heating tube).

Alternatively, a spray drying method may be used. In this case, agelatin solution is sprayed from an atomizer or a nozzle into a heateddrying chamber.

From the viewpoint of further enhancing the above-described effects, thedifference between the temperature of an atmosphere in the heating tubeor the drying chamber and the temperature of a dropped gelatin solutionis preferably 235° C. or less. Further, from the viewpoint ofefficiently and stably obtaining gelatin particles, the temperaturedifference is preferably 20° C. or more but 200° C. or less, morepreferably 20° C. or more but 100° C. or less. Particularly, thetemperature of an atmosphere in the heating tube or the drying chamberis preferably higher than the temperature of droplets by 20° C. or morebut 80° C. or less. The temperature of droplets is preferably 15° C. orhigher but 80° C. or lower, more preferably 20° C. or higher but 50° C.or lower. The temperature of an atmosphere in the heating tube or thedrying chamber is preferably 40° C. or higher but 250° C. or lower, morepreferably 40° C. or higher but 150° C. or lower.

From the viewpoint of making gelatin particles whose particle diameterafter swelling treatment is 1.0 nm or more but 5.0 μm or less morelikely to be produced, the gelatin content of the gelatin solution ispreferably 1.00×10⁻⁸ vol % or more but 60 vol % or less, more preferably1.00×10⁻⁷ vol % or more but 50 vol % or less, even more preferably1.00×10⁻⁷ vol % or more but 20 vol % or less.

From the viewpoint of making gelatin particles having a swelling degreeof 1.0 or more but 10.0 or less more likely to be produced, gelatinparticles are preferably crosslinked.

The crosslinking of gelatin particles may be crosslinking achieved usingthe above-mentioned crosslinking agent or self-crosslinking achieved byapplication of heat or by irradiation with electron beams or ultravioletrays.

When easy-uptake gelatin particles carrying a reagent or the like areproduced, a gelatin solution obtained by previously mixing gelatin and areagent or the like may be used to form the gelatin into particles.

2. Cell

This embodiment relates to a cell containing easy-uptake gelatinparticles inside the cell membrane and a method for producing such acell.

2-1. Cell

The cell according to this embodiment is a cell containing easy-uptakegelatin particles inside the cell membrane (hereinafter, also referredto as “gelatin particle-containing cell”).

The phrase “containing gelatin particles inside the cell membrane” meansthat in an image of a cell taken by a transmission electron microscope(TEM), gelatin particles are observed inside the cell membrane. Theuptake of gelatin particles into cells can be confirmed in the followingmanner. For example, when gelatin particles contain a contrast medium,the determination as to whether or not the gelatin particles containinga contrast medium have been taken up into cells can be made by stainingand microscopically observing the contrast medium. When not containing acontrast medium, gelatin particles may be previously fluorescentlylabeled. In this case, the determination as to whether or not thefluorescently labeled-gelatin particles have been taken up into cellscan be made using a confocal microscope. The fluorescent labeling ofgelatin particles can be performed using, as a substrate, FITC-gelatinprepared by, for example, mixing equal amounts of a solution labeledwith fluorescein isothiocyanate (FITC) (e.g., a 10 mM acetic acidsolution of FITC-collagen manufactured by Cosmo Bio Co., Ltd.), 0.4 Msodium chloride, 0.04% (W/V) sodium azide, and a 50 mM tris-HCl buffercontaining 10 mM calcium chloride (pH 7.5) and then heating the mixtureat 60° C. for 30 minutes.

The easy-uptake gelatin particles contained in cells preferably carry acontrast medium, especially a contrast medium for MRI. Such cells areproduced by a method described later in which easy-uptake gelatinparticles are taken up into cells by cell's own action. Therefore, theactivity of the cells can be non-destructively examined by observing thepresence or absence of the contrast medium in the cells.

Examples of cells capable of containing gelatin particles inside thecell membrane include known cells including cells derived frombiological samples or specimens extracted from various organs such asbone marrow, heart, lung, liver, kidney, pancreas, spleen, intestinaltract, small intestine, cardiac valve, skin, blood vessel, cornea,eyeball, dura mater, bone, trachea, and auditory ossicles,commercially-available established cell lines, stem cells such as skinstem cells, epidermal keratinocyte stem cells, retinal stem cells,retinal epithelial stem cells, cartilage stem cells, hair follicle stemcells, muscle stem cells, osteoprogenitor stem cells, preadipocyte stemcells, hematopoietic stem cells, nerve stem cells, hepatic stem cells,pancreatic stem cells, ectodermal stem cells, mesodermal stem cells,endodermal stem cells, mesenchymal stem cells, ES cells, and iPS cells,and cells differentiated from these stem cells.

Among these cells, when cells, especially stem cells and cellsdifferentiated from stem cells, to be transplanted into a patient incellular regenerative medicine contain easy-uptake gelatin particlescarrying a contrast medium, especially a contrast medium for MRI, thedetermination as to whether or not the gelatin particle-containing cellshave been colonized in a transplantation site can be made withoutreoperation by observing the contrast medium in the transplantation siteafter transplantation into a patient. Therefore, it is considered thatthese cells containing gelatin particles carrying a contrast medium forMRI can reduce the physical, mental, financial, and temporal burden onpatients who receive regenerative medicine treatment and improve thequality of life (QOL) of the patients.

2-2. Method for producing cell

The gelatin particle-containing cell can be produced by introducingeasy-uptake gelatin particles into the cell mentioned above. Examples ofa method for introducing gelatin particles into a cell include a methodin which gelatin particles and a cell are added to a liquid to allow thegelatin particles to be taken up into the cell by cell's own action suchas endocytosis and a method in which gelatin particles are introducedinto a cell by external operation. Examples of the method in whichgelatin particles are taken up into a cell by cell's own action includea method in which gelatin particles and cells are stirred in a liquidand a method in which cells are cultured in a cell culture mediumcontaining gelatin particles. It is to be noted that the above-describedeasy-uptake gelatin particles are very efficiently taken up by cellsthemselves, and therefore it is not particularly necessary to performoperation for forming a complex with another component to promote uptakeinto cells. From the viewpoint of minimizing a reduction in the activityof cells, among the above methods, a method is preferred in which cellsare mixed with easy-uptake gelatin particles in a liquid and cultured.Examples of the method in which gelatin particles are introduced into acell by external operation include an electroporation method and amicroinjection method. Among them, from the viewpoint of preventing areduction in the activity of cells during the introduction of gelatinparticles, a method in which gelatin particles are introduced into acell by cell's own action is preferred, and a method in which gelatinparticles are taken up into a cell without forming the above-describedcomplex is more preferred.

The liquid to which gelatin particles and cells are added may be a cellculture medium. The cell culture medium may be a known buffer orphysiological saline, and examples thereof include Hanks' Balanced SaltSolution (HBSS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), and another known phosphate buffered saline (PBS).

From the viewpoint of enhancing the activity of cells to facilitate theuptake of gelatin particles into the cells by cell's own action, thetemperature of the cell culture medium during stirring is preferably 15°C. or higher but 50° C. or lower, more preferably 35° C. or higher but45° C. or lower.

When gelatin particles are introduced into the inside of the cellmembrane by cell's own action, introduction of gelatin particles may bepromoted by, for example, shaking the cell culture medium containing thegelatin particles and the cells.

It is considered that when gelatin particles are introduced into cellsby cell's own action, high-activity cells are more likely to take upgelatin particles, but low-activity cells are less likely to take upgelatin particles. Therefore, the activity of cells can benon-destructively examined by adding gelatin particles carrying acontrast medium and cells to a liquid and, if necessary, shaking theliquid, and then by observing the presence or absence of the contrastmedium in the cells.

EXAMPLES

Hereinbelow, specific examples of the present invention will bedescribed. It is to be noted that the scope of the present inventionshould not be construed as being limited to these examples.

1. Production of Gelatin Particles

1-1. Preparation of Raw Material Solutions

Gelatin (G-2613P manufactured by Nitta Gelatin Inc.), pure water, Fe2O3powder (3310DX (α-Fe₂O₃) manufactured by COREFRONT Corporation) weremixed to prepare a raw material solution containing the gelatin and theFe2O3 powder in a volume ratio of 10:1 (gelatin:Fe2O3 powder=10:1). Theamounts of the gelatin and the Fe2O3 powder were adjusted so that theraw material solution had a gelatin concentration and a Fe₂O₃concentration shown in Table 1.

1-2. Production of Gelatin Particles by Inkjet Method

An air flow of 3 L/min was blown into a heating tube heated to 100° C.in a vertical direction from the top toward the bottom of the heatingtube. Droplets of 4 pL or 42 pL of the raw material solution heated to40° C. were dropped from an inkjet head (512S manufactured by KonicaMinolta Co., Ltd.) into the air flow at an ejecting frequency of 5 kHzand landed on a hydrophilically-treated polytetrafluoroethylene resin(PTFE) filter (Millipore, 0.45 mesh, manufactured by Merck (NihonMillipore K. K.)) located 200 cm below the inkjet head. The dropping wasperformed for 5 hours, and then gelatin particles on the filter werecollected.

1-3. Production of Gelatin Particles by Spray Drying Method

The raw material solution was sprayed from a two-fluid-type nozzle of aspray dryer (SPRAY BOY manufactured by PRECI Co., Ltd.) into a dryingchamber heated to 200° C. at a rate of 1 kg/h, and gelatin particleswere collected from the bottom of the drying chamber.

1-4. Crosslinking

The gelatin particles produced above were heated in a vacuum heatingfurnace (Vacuum Oven ADP200 manufactured by Yamato Scientific Co., Ltd.)at 160° C. for a predetermined time shown in Table 1. In this way,Gelatin Particles 1 to 15 were obtained.

Table 1 shows the volume percentages of gelatin and Fe₂O₃ in each of theraw material solutions used for producing Gelatin Particles 1 to 16, aparticle production method, the quantity of each droplet when particleswere produced by an inkjet method, the temperature in a system duringparticle production, a liquid feed rate, and the temperature and time ofheating in a heating furnace.

TABLE 1 Production of Gelatin Particles 1 to 16 Particle productionHeating (Crosslinking) Raw material solution Quantity Temperature LiquidHeating Heating Gelatin Fe₂O₃ of droplet in system feed rate temperaturetime (vol %) (vol %) Method (pL) (° C.) (ml/h) (° C.) (min) GelatinParticle 1 4.88 × 10⁻⁷ 4.88 × 10⁻⁸ Inkjet 4 100 100 160 30 GelatinParticle 2 1.45 × 10⁻⁷ 1.45 × 10⁻⁸ Inkjet 4 100 100 160 1 GelatinParticle 3 1.81 × 10⁻⁵ 1.81 × 10⁻⁶ Inkjet 4 100 100 160 2 GelatinParticle 4 6.10 × 10⁻² 6.10 × 10⁻³ Inkjet 4 100 100 160 0.03 GelatinParticle 5 1.65 0.165 Inkjet 4 100 100 160 30 Gelatin Particle 6 6.10 ×10⁻² 6.10 × 10⁻³ Inkjet 4 100 100 160 15 Gelatin Particle 7 9.26 × 10⁻³9.26 × 10⁻⁴ Inkjet 4 100 100 160 5 Gelatin Particle 8 2.26 × 10⁻³ 2.26 ×10⁻⁴ Inkjet 4 100 100 160 10 Gelatin Particle 9 2.26 × 10⁻³ 2.26 × 10⁻⁴Spray drying — 200 1000 160 15 Gelatin Particle 10 2.26 × 10⁻³ 2.26 ×10⁻⁴ Spray drying — 200 1000 160 15 Gelatin Particle 11 2.40 × 10⁻³ 2.40× 10⁻⁴ Spray drying 4 200 100 160 15 Gelatin Particle 12 1.81 × 10⁻²1.81 × 10⁻³ Inkjet 4 100 100 160 0.01 Gelatin Particle 13 18.1 18.1Inkjet 4 100 100 160 30 Gelatin Particle 14 1.81 × 10⁻⁵ 1.81 × 10⁻⁶Inkjet 4 100 100 160 1.5 Gelatin Particle 15 46.5 4.65 Inkjet 42 100 100160 50 Gelatin Particle 16 1.75 0.175 Inkjet 4 100 100 160 30

2. Measurement of Gelatin Particles

2-1. Major-Axis Length, Average Particle Diameter, and Aspect Ratio ofDried Gelatin Particles

Gelatin particles of each of Gelatin Particles 1 to 16 produced abovewere imaged with a scanning electron microscope (SEM). The taken imagewas analyzed using Mac-View that is particle size distribution analysissoftware manufactured by Mountech Co., Ltd. to measure the minor-axislengths and the major-axis lengths of randomly-selected 20 gelatinparticles, and the averages thereof were defined as the minor-axislength and the major-axis length (a) of each of dried Gelatin Particles1 to 16. The average of the minor-axis length and the major-axis lengthof each of the randomly-selected 20 dried gelatin particles wasdetermined as the particle diameter of each of the 20 gelatin particles,and the average of the thus determined particle diameters of the 20gelatin particles was defined as the average particle diameter of eachof dried Gelatin Particles 1 to 16. Further, the major-axis length ofeach of the randomly-selected 20 dried gelatin particles was divided bythe minor-axis length of each of the randomly-selected 20 dried gelatinparticles to determine the aspect ratio of each of the 20 gelatinparticles, and the average of the thus determined aspect ratios of the20 gelatin particles was defined as the aspect ratio of each of driedGelatin Particles 1 to 16.

2-2. Swelling Treatment and Major-Axis Length and Average ParticleDiameter of Gelatin Particles after Swelling Treatment

First, 0.1 g of gelatin particles of each of Gelatin Particles 1 to 16produced above were immersed and dispersed in 100 mL of pure water at40° C. and were allowed to stand for 60 minutes (swelling treatment).Then, the gelatin particles were imaged with a scanning electronmicroscope (SEM). The taken image was analyzed using Mac-View that isparticle size distribution analysis software manufactured by MountechCo., Ltd. to measure the minor-axis length and the major-axis length ofeach of randomly-selected 20 gelatin particles, and the average of thethus measured minor-axis length and major-axis length was determined asthe particle diameter of each of the 20 gelatin particles. The averageof the thus determined particle diameters of the 20 gelatin particleswas defined as the average particle diameter of each of Gelatinparticles 1 to 16 after swelling treatment.

2-3. Swelling Degree

The average particle diameter of gelatin particles after swellingtreatment was divided by the average particle diameter of dried gelatinparticles to determine the swelling degree of each of Gelatin Particles1 to 16.

Table 2 shows the major-axis lengths, average particle diameters, andaspect ratios of dried Gelatin Particles 1 to 16 and the swellingdegrees of Gelatin Particles 1 to 16.

TABLE 2 Major-Axis Lengths, Average Particle Diameters, and AspectRatios of Dried Gelatin Particles 1 to 16 and Swelling Degrees ofGelatin Particles 1 to 16 Under dry conditions After swelling treatmentAverage Average Minor-axis Major-axis particle Major-axis particleSwelling length length (a) diameter Aspect length (b) diameter degree(μm) (μm) (μm) ratio (μm) (μm) (b/a) Gelatin Particle 1 0.029 0.031 0.031.07 0.034 0.03 1.1 Gelatin Particle 2 0.020 0.020 0.02 1.00 0.080 0.084.0 Gelatin Particle 3 0.098 0.102 0.10 1.04 0.918 0.90 9.0 GelatinParticle 4 1.463 1.537 1.50 1.05 4.611 4.50 3.0 Gelatin Particle 5 4.3904.610 4.50 1.05 5.071 4.95 1.1 Gelatin Particle 6 1.463 1.537 1.50 1.052.306 2.25 1.5 Gelatin Particle 7 0.780 0.820 0.80 1.05 1.640 1.60 2.0Gelatin Particle 8 0.488 0.512 0.50 1.05 1.024 1.00 2.0 Gelatin Particle9 0.417 0.583 0.50 1.49 0.875 0.75 1.5 Gelatin Particle 10 0.357 0.6430.50 1.80 0.965 0.75 1.5 Gelatin Particle 11 0.498 0.522 0.51 1.05 1.0441.02 2.0 Gelatin Particle 12 0.976 1.024 1.00 1.49 12.288 12.00 12.0Gelatin Particle 13 9.756 10.244 10.00 1.05 51.220 50.00 5.0 GelatinParticle 14 0.098 0.102 0.10 1.04 1.530 1.50 15.0 Gelatin Particle 1529.268 30.732 30.00 1.05 61.464 60.00 2.0 Gelatin Particle 16 4.5454.636 4.59 1.02 5.100 5.05 1.1

3. Introduction into Cells and Evaluations

A cell culture medium was used which was prepared by adding 50 mL offetal bovne serum to 500 mL of a cell culture medium MEM Alpha basic(1X) manufacture by Life Technologies. One milligram of each of GelatinParticles 1 to 16 was added to 3 mL of the cell culture medium, andmouse osteoblast-derived cells (MC3T3E1) were added at 6000 cells/mL.The cell culture medium after cell addition was incubated at 40° C. for24 hours. In this way, 16 evaluation samples were prepared.

Then, part of each of the cell suspensions was taken out, and thedetermination as to whether or not gelatin taken up inside the cellmembrane could be confirmed was made in the following manner.

(Staining of Cells and Fe)

First, 1 ml of 1% paraformaldehyde was added to the cultured cells toperform cell immobilization treatment. Then, 1 ml of an Fe stainingsolution having the following composition was added to stain Fe.Further, 1 mL of a nuclear staining solution adjusted to the followingconcentration was added to stain the cells.

(Composition of Fe Staining Solution)

Equal volumes of the following two solutions were mixed to prepare an Festaining solution.

-   -   20 vol % HCL (5-fold dilution of concentrated hydrochloric acid)    -   10 mass % aqueous K₄(Fe(CN₆)) solution (100 mg/mL)

(Composition of Nuclear Staining Solution)

Five parts by mass of ammonium sulfate and 0.1 parts by mass of Nuclearfast red were mixed with 100 parts by mass of distilled water to preparea nuclear staining solution.

(Counting of Number of Cells That Have Taken up Fe)

The stained cells were observed with an optical microscope to evaluatewhether stained Fe was contained in randomly-selected 20 cells.

In the cases of the cell suspensions of Gelatin Particles 1 to 11 whoseswelling degree was 1 or more but 10 or less and average particlediameter after swelling treatment was 1.0 nm or more but 5.0 μm or less,out of the 20 cells, 10% or more of the cells (2 or more cells) wereconfirmed to have taken up gelatin inside the cell membrane.

On the other hand, in the cases of the cell suspensions of GelatinParticles 12, 13, 15, and 16 whose average particle diameter afterswelling treatment was 5.0 μm or more, out of the 20 cells, less than10% of the cells (less than 2 cells) took up the gelatin particlesinside the cell membrane. The reason for this is considered to be thatcells recognized the gelatin particles as foreign matter due to theirlarge average particle diameter after swelling treatment, and thereforethe gelatin particles were hard to be taken up into cells by cell's ownaction.

Further, in the cases of the cell suspensions of Gelatin Particle 14whose swelling degree was larger than 10, out of the 20 cells, less than10% of the cells (less than 2 cells) took up the gelatin particlesinside the cell membrane. The reason for this is considered to be thatsince the gelatin particles absorbed a large amount of water andaggregated, cells recognized the gelatin particles as foreign matter dueto their large apparent particle diameter, and therefore the gelatinparticles were hard to be taken up into cells by cell's own action.

It is to be noted that Gelatin Particles 1 to 8 produced by an inkjetmethod generally had a small aspect ratio. For example, Gelatin Particle8 had a smaller aspect ratio than Gelatin Particles 9 and 10 having thesame particle diameter as Gelatin Particle 8 and produced by a spraydrying method.

This application is based upon and claims the benefit of priority fromprior Japanese Patent Application No. 2015-254950 filed on Dec. 25, 2015in Japan, the specification and claims of which are incorporated hereinby reference.

INDUSTRIAL APPLICABILITY

The gelatin particles according to the present invention can contain,for example, a contrast medium for MRI and can be introduced into cellsfor transplantation used in regenerative medicine. Such cells areproduced by allowing the gelatin particles to be taken up by cell's ownaction, and the activity of the cells can be non-destructively examinedby observing the presence or absence of the contrast medium in the cellsby MR imaging Therefore, it is considered that the use of the gelatinparticles according to the present invention makes it possible to reducethe wastage rate of cells used in regenerative medicine and increase theuse efficiency of the cells. Further, when such cells are transplanted,a transplantation site can be observed not by reoperation but by MRimaging to determine whether or not the cells have been colonized in thetransplantation site. Therefore, it is considered that the gelatinparticles according to the present invention can reduce the physical,mental, financial, and temporal burdens on patients and improve thequality of life (QOL) of the patients.

1. Gelatin particles, wherein when a major-axis length of dried gelatinparticles is defined as a and a major-axis length of gelatin particlesafter swelling treatment obtained by immersing the dried gelatinparticles in water at 40° C. under an atmospheric pressure for 60minutes is defined as b, swelling degree represented by b/a is 1.0 ormore but 10.0 or less, and wherein the gelatin particles after swellingtreatment have a particle diameter of 1.0 nm or more but 5.0 μm or less.2. The gelatin particles according to claim 1, wherein the dried gelatinparticles have an aspect ratio of 1.0 or more but 1.4 or less.
 3. Thegelatin particles according to claim 1, wherein the major-axis length bof the gelatin particles after swelling treatment is 2.0 μm or less. 4.The gelatin particles according to claim 1, further comprising acontrast medium.
 5. The gelatin particles according to claim 1, whereinthe gelatin has been crosslinked.
 6. A method for producing gelatinparticles, comprising discharging droplets of a liquid containing meltedgelatin into an atmosphere in a heating tube or a drying chamber whosedifference in temperature from the droplets is 235° C. or less anddrying the droplets to obtain gelatin particles.
 7. The method forproducing gelatin particles according to claim 6, wherein the dropletsare discharged from a nozzle of an inkjet head.
 8. A gelatinparticle-containing cell, wherein the gelatin particles according toclaim 1 are contained inside a cell membrane.
 9. A method for producinga gelatin particle-containing cell, comprising adding the gelatinparticles according to claim 1 and a cell to a liquid to allow thegelatin particles to be taken up inside a cell membrane of the cell byaction of the cell.
 10. The gelatin particles according to claim 2,wherein the major-axis length b of the gelatin particles after swellingtreatment is 2.0 μm or less.
 11. The gelatin particles according toclaim 2, further comprising a contrast medium.
 12. The gelatin particlesaccording to claim 2, wherein the gelatin has been crosslinked.
 13. Agelatin particle-containing cell, wherein the gelatin particlesaccording to claim 2 are contained inside a cell membrane.
 14. A methodfor producing a gelatin particle-containing cell, comprising adding thegelatin particles according to claim 2 and a cell to a liquid to allowthe gelatin particles to be taken up inside a cell membrane of the cellby action of the cell.
 15. The gelatin particles according to claim 3,further comprising a contrast medium.
 16. The gelatin particlesaccording to claim 3, wherein the gelatin has been crosslinked.
 17. Agelatin particle-containing cell, wherein the gelatin particlesaccording to claim 3 are contained inside a cell membrane.
 18. A methodfor producing a gelatin particle-containing cell, comprising adding thegelatin particles according to claim 3 and a cell to a liquid to allowthe gelatin particles to be taken up inside a cell membrane of the cellby action of the cell.
 19. The gelatin particles according to claim 4,wherein the gelatin has been crosslinked.
 20. A gelatinparticle-containing cell, wherein the gelatin particles according toclaim 4 are contained inside a cell mem